Elevated levels of total and low-density lipoprotein (LDL) cholesterol are associated with increased risk for atherosclerosis. Individuals within the human population exhibit a large range of cholesterol levels, likely determined by the interplay between environmental and genetic factors. Currently known genetic variations account for only a fraction of the total variance of cholesterol levels, suggesting that novel pathways and genes remain to be identified. Using positional cloning, we have identified the Diet1 gene from an inbred mouse strain that is resistant to diet-induced hypercholesterolemia and atherosclerosis. Diet1 encodes a novel protein characterized by repeating MAM and LDL receptor type A domains, and is expressed predominantly in the small intestine. The Diet1 protein sequence is highly conserved between mouse and humans. We therefore hypothesize that the human DIET1 gene is an excellent candidate gene for effects on cholesterol levels and related traits in humans. We propose to characterize Diet1 function at the molecular, cellular and physiological levels. The specific aims are: (1) Determine the cellular role of Diet1 in lipid metabolism. Our studies indicate that Diet1 is expressed in the small intestinal epithelial cells, and we hypothesize that it functions in intracellular bile acid transport. We will determine the cellular compartment(s) in which Diet1 functions, characterize the potential role of Diet1 in cellular lipid transport, and investigate the regulation of DIET1 gene expression. (2) Determine the physiological role of Diet1 in the regulation of cholesterol homeostasis. Diet1 deficient mice exhibit enhanced bile acid excretion, increased bile acid synthesis, and impaired induction of fibroblast growth factor 15 (FGF15), a key intestinal signal for the regulation of hepatic bile acid synthesis. We will test the hypothesis that the alterations in cholesterol homeostasis in Diet1 deficient mice can be attributed to impaired FGF15 regulation by FGF15 replacement in vivo. We will also determine whether enhanced Diet1 expression leads to altered enterohepatic signaling or altered cholesterol homeostasis using a Diet1 transgenic mouse. (3) Determine the mechanism underlying enhanced adaptive thermogenesis in Diet1 deficient mice. Diet1 deficient mice exhibit enhanced basal energy expenditure and adaptive thermogenesis. We hypothesize that increased circulating bile acid levels resulting from Diet1 deficiency stimulate increased fatty acid fuel availability mediated by effects on FGF21. We will test this mechanism by studies in isolated brown adipocytes, and by FGF21 administration in wild-type mice. (4) Identify common and rare variants in human DIET1 and determine association with cholesterol levels. We hypothesize that common or rare DIET1 genetic variants influence cholesterol levels in the human population. We will resequence the DIET1 coding exons from individuals with extreme high and low bile acid and LDL-C levels in a population-based sample of 8000 individuals. We will identify common and rare variants that lead to potential changes in protein function, and test these using functional assays. PUBLIC HEALTH RELEVANCE: We have identified a mutation in a novel gene-Diet1-that confers resistance to high blood cholesterol levels and atherosclerosis in the mouse. Here we will determine how Diet1 functions in the intestine to regulate bile acid synthesis and cholesterol levels using mouse models, and will investigate whether sequence variations in the human Diet1 gene influence cholesterol levels and cardiovascular disease risk in the human population. Results will further elucidate the mechanisms that control cholesterol homeostasis, and may suggest new strategies for protection or treatment of hypercholesterolemia and related disorders.